Phosphorus and Mineral Concentrations in Whole Grain and Milled Low Phytic Acid (ipa) 1-1 Rice

Cereal Chem. 82(5):5 17-522 Phytie acid iins'o-inositolI .2.3.4.5.6-hexaki.sphosphate) is the most abundant lorni of phosphorus (P) in cereal grains and is important to grain nutritional qualit y . In [nature rice ( Orv:a rota L) erains, the bulk Of phytic acid P is found in the germ and aleurone layer. deposited primarily as a mixed K/Mg salt. Phosphorus components and minerals were measured in whole grain produced by either the rice (Orva sath'a L.) cv. Kayhonnet (the nonmutant control) or the low phytic acid I-I ((pa 1I) mutant, and in these grains when milled to di lierent degrees (1 0, 12. 17. 20. 22. and 25'%. "AN I. Phytie acid P is reduced b y 42-45i in (pal I whole grain as compared with Ka y bonnet. but these whole grains had similar leseis of total P. Ca. Fe. K. Mg. Mn. and Zn. In both genot ypes, the concentration of phytic acid P. total P. Ca. Fe, K. Mg. and Mn in the milled products was reduced by 60-90, as compared with whole grain. However, a trend was observed for higher I 25-407 I total P. K. and Mg concentrations in (pa I I milled products as compared with Kavhonnet milled products. The reduction in whole grain phy tic acid P in nec (pa I I is accompanied by a 5to 10-fold increase in grain inorganic P. and this increase was observed in both whole grain and milled products. Phytic acid P was also reduced by 45 in bran obtained Irons 1a I I grain, and this was accompanied by a 10-fold increase in inorganic P. Milling had no apparent effect on Zn concentration. Therefore, while the block in the acctimui latiott of phvtic acid in (pal I seed has little effect on whole grain total P and mineral concentration, it greatly alters the chemistry of these seed constituents, and to a lesser but detectable extent, alters their distribution between germ, central endosperin, and aleurone. These studies suggest that development of a low phytate rice might improve the nutritional qualit of whole grain, milled rice and the bran produced during IllillifIL.. Phyhic acid (mvo-inositolI .2.3.4.5.6-hexakisphosphate) is the most abundant form of phosphorus (P) in seeds, typically representing 65804 of seed total P. and from one to several percent of seed dry weight (Lott et al 2000). It also represents the most abundant lorm of ,nvo-inosiloi phosphates (Ins phosphates) in seeds (Rahoy 2003). Seed-derived dietary phytic acid may have a positive role as all and anticancer agent (Graf and Eaton 1993). However, humans and other nont'Litninant animals such as poultry, swine, and fish excrete most of the phytic acid they consume. In the context of livestock production, this mostly represents a P-management issue, both in terms of supplying adequate nutrient P foroptimal livestock produtctivitv and in managing the disposal of waste P. In the context of liutman nutrition, the primary concern is the impact of dietary phytic acid oil cation retention. particularly with reference to iron and zinc nutrition. Phytic acid is an effective chelator of minerals such as iron, calciuin. and zinc (Cilliers and van Niekerk 1986). The fact that phytic acid is poorly digested by humans and is an effective chelator of minerals call ii negative impact oil retention and utilization of minerals. Chronic consumption of phytic acid by populations dependent on cereals and legumes. which are rich sources ol phytic acid, call to mineral deficiency (Erdman 1981). In several developing countries, the consumption of rice provides the majority of the calories consumed. In many of these countries, mineral deficiencies are common (Brown and Solomons 1991). In mature rice (()rva saliva I,.) and wheat (Tritit-wn ae.s'tit'om L.) grains, the bulk of whole grain phytic acid and minerals are found in the germ (embryo arid scutetlLim) and aleurone layer (O'Dell et a! 1972). Therefore, the removal of the outer layers of LSDA-ARS. Dale ttiunpc'rs National Rice Research Center. EQ. Box 1090, Siuttgai't, AR 72 160. Names are necessary to report tdcivally on available data: howeser, the USDA neither guarantees nor warrants the standard or the product. and the use of the name by the USDA implies no approval of the product t o the exclusion of others that may also he suitable. 2 Corresponding auihor. Phone: 1-870-672-9300 (Ext. 227). Fax 1-870-673-7581. E-mail address: rhryantspa.ars.uisda.gov USDA-.ARS, Small Grains and Potato Germplasns Research Facility, Aberdeen, ID 83210. Current address: BASE Corporation. Research Triangle Pat k, NC 27709. DOl: 10.1094/CC-82-0517 This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. AACC International, Inc., 2005. the rice and wheat grain thi'ough milling should reduce both milled grain phytic acid and minerals. While brown rice contains many beneficial nutrients. the presence of phytic acid might i'ecluce its value in terms oh mineral nutritional health. Ogawa ci al (1979) demonstrated that early in rice grain development P. K. and Mg are evenly distributed hhut'oughouit the central endosperm, aleui'one la yer, and germ tissues. As grains approach mattirity, progressively mote oh' the total P. K. and Mg is concentrated in the germ and aleurone, most of which is deposited as a mixed phytin salt of K and Mg. packaged into discreet inclusions referred to as glohoids (O gawa et al 1979: Liu et al 2004). Therefore, the ability of seeds to localize phytic acid synthesis might be part of the tuicehanism by which minerals such as K and Mg are also concentrated in specific tissues. Low-phytate variants of maize (Zea ;navs L.). barley (Hard coin m'ulgarc L.), rice, and wheat have been developed through the isolation of low phytic acid ((pci) mutants in each species (Larson et al 2000: Raboy et al 2000: Raboy 2001: Dorsch et ill 2003: Gull icri et al 2004). The first rice low-phytate iuuutant was isolated (Larson ci al 2000) by screening a population of mutations induced in the cultivar Ka ybonnet by y-irradiation. Inheritance analysis indicated that the reduction in whole grain phytic acid P. m45 17c as compared with the nonmutant Kayhonnet control, was due to the inheritance of' a single-gene mutation, rice low phytic acid 1(1pa 1-I ) (Larson et al 2000). Normal Kaybonnet whole grains contained w2.23 mg of phytic acid Pig. whereas (pal I grains contained w1.37 mg of phytic acid Pig. Reduced phytie acid P in rice (pal I seeds did not appear to be due to a reduction in seed total P (m3.1 2 mg of total PA, in Kayhonnet compared with 3.55 mng of total Pig in (pa I I). Instead, the reduction in phytic acid P was largely matched, in ternus of R by all iii whole grain inorganic P. I'i'orn 0.14 nug of inorganic P/g in Ktm yboiunet to 1.13 mg of inorganic P/g in (pal -1. Liu et al (2004) conducted the first study of the distribution and deposition of P and minerals in rice 11)(1 1-1 grains. Whole grains of Kayhonnet and (pal I were dissected into two fractions: embryo. consisting of the embryo and scuitellutus: rest-of-grain, eomusistimug of the central, starchy endospermn, and aleutrone layer. Analyses of the mineral concentrations in these two fractions found no large differences hetweemu Ka ybonnet (muormnal or wild-type control) and (pa h-i in the whole grain total amuuoumnt or distribution total t t l P. K. Mg, Ca. Fe. or Zn (Liu ci at 2004). However, the cxperimuuenmtal Vol. 82, No. 5, 2005 517 design used could not detect differences in distribution between central endosperni and aleurone layer because both were contained within the rest-of-grain fraction. If rice cultivars with the low phytatc trait are produced, knowledge concerning the impact, if any. of the ipa mutation on the concentration of P components and mineral cations in iii ill in g products is of practical importance. In research described here, rice grain produced by Kayhonnet and the ij,alI mutant (Rutger et at 2003) were milled to different degrees, and the concentration of P components and minerals were assayed in the milled product. Milling removes the outer portions of the rice grain, including both germ and aleurone, producing two types of milled products enriched either in the central, starchy endosperm (white rice) or in the germ and aleurone (bran). Therefore, analyses of P and minerals in such products Should also provide a test of the hypothesis that the localization of phytic acid synthesis in the cereal grain has a functional role in P and mineral localization. MATERIALS AND METHODS The rice cv. Kaybonnet and the low phytic acid 1-1 (ipal-1 mutant were grown at the Dale Bumpers National Rice Research Center, Stuttgart. AR, in each of three years (2000. 2001. and 2002). Kayhonnet is the cultivar or genetic background from which the ipa I -I mutant was isolated and serves here as the normal, noninutant. or wild-type control. Grains were dehufled in a sample sheller (THU 35A1, Satake Engineerin g , Tokyo, Japan). The brown rice was subjected to different degrees of milling using a McGill #2 mill (Grain Machinery Manufacturing. Miami, FL). In grains harvested in 2001. milling products were obtained where 0% (whole grain). 10. 12. 17. 20. 22. or 25% of tissue was removed. In grains harvested in 2000 and 2002, milling products were obtained where OC/ (whole grain). 10. 15. or 20% was removed. In addition, in 2000 and 2002. bran fractions, defined here as representing that portion of the grain removed by 10% milling, were obtained. Each sample was ground (Cyclotech grinder. Foss North America, Eden Prairie, MN) equipped with a 0.5-mm screen, and moistures were determined using Approved Method 44-I5A (AACC International 2000). Total P. phytic acid P. and inorganic P of the whole grain and milled products were measured using methods described earlier (Rahoy et at 2000: Dorsch et at 2003). Briefl y, samples of mature grain or milled products were dried for 48 hr at 60°C. These were then milled to pass through a 20-mm screen and stored in a desiccator until analysis. Total P was determined after wet-ashing of aliquots of tissue (150 mg) and colorimetric assay of P in the digests (Chen et at 1956). Inorganic P was determined colorinietrically after extraction of tissue samples (0.5 of in 12.5% (w/v) TCA and 25 mM MgCt. The ferric-precipitation method was used to determine phytate P (Dorsch et a] 2003). Aliquots of tissue (0.5 g) were extracted in 0.4M HCI and 0.7M NaSO 4 . Phytic acid P was then obtained as a ferric precipitate, wet-ashed and assayed for P as in the total P analysis. All P-containing fractions are expressed as their P (atomic weight 3!) content to facilitate comparisons. Phytic acid P can he converted to units of phytic acid (MW 660) by multiplying by the conversion factor 3.548. Mineral cation composition of whole grain and milled products were determined by the Universit y of Idaho Analytical Sciences Laboratory. Holm Research Center in Moscow. ID. Flour samples (1 g) were digested with nitric acid, and mineral concentrations in the digests were determined using a inductively coupled plasmaoptical emission spectrometer (Perkin-Elmer Optima 3200 ICPOES. University of Idaho, Moscow, ID). Analysis of variance (ANOVA) was conducted to determine F values useful in testing the effect on P and mineral concentrations of genotype, milling degree, and the interaction of genotype and milling degree. The general linear model (GLM) procedure (SAS Institute. Cary. NC was used. RESULTS AND DISCUSSION The first analysis of P components and mineral cations in milled products 01' 1/?(1 1-1 grain was conducted using materials produced in 2001 (Table I). The concentrations of total P. phytic acid P. and inorganic P in Kaybonnet and Ipal I whole grain (0 1 c milling) were similar to that initially reported by Larson et at (2000). Similar to the results of Larson et a] (2000), little or no difference in whole g rain total P was observed between these two genotypes, while whole grain phytic acid P was reduced 43% in Ipa I I . as compared with Kaybonnet, and this reduction was accompanied by a similar increase in whole grain inorganic P. In both genotypes. removal of outer portions of the grain through milling produced products with total P concentrations reduced by 49_7fie/r, and pliytic acid P levels were reduced by 63-92%, as compared with whole grains (Table 1). However, concentrations of total P in the milled products of ipa I I grain were I 6-48% higher than those TABLE I Phosphorus Concentrations in Rice cv. Ka ybonnet and Low Ph ytic Acid (ipa) 1-1 Cram When Milled to Different Degrees, Production Year 2001